This invention relates to wild-type isoforms and mutations of Ikaros, and to nucleic acid sequences encoding Ikaros, useful in the diagnosis and treatment of hematologic malignancy, particularly lymphoid malignancy, including stem cell leukemia and T-cell and B-cell acute lymphoblastic leukemia (ALL).
Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children Leukemic clones in ALL patients are thought to originate from normal lymphocyte precursors arrested at various stages of T- or B-lymphocyte development hence, any critical regulatory network that controls normal lymphocyte development is a potential target for a leukemogenic event.
One such regulatory network vital for normal lymphopoiesis involves Ikaros, a member of the Kruppel family xe2x80x9czinc fingerxe2x80x9d DNA-binding proteins. Ikaros acts as an evolutionarily conserved xe2x80x9cmaster switchxe2x80x9d of hematopoiesis that dictates the transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation.1 Programmed expression and function of the Ikaros gene is tightly controlled by alternative splicing of Ikaros pre-mRNA which results in production of eight different Ikaros isoforms. All eight Ikaros isoforms (Ik-1, Ik-2, Ik-3, Ik-4, Ik-5, Ik-6, Ik-7, and Ik-8) share a common carboxy(C)-terminal domain containing a transcription activation motif and two zinc finger motifs that are required for hetero- and homodimerization among the Ikaros isoforms and for interactions with other proteins.
1Georgopolous et al., 1994, Cell, 79:143-156; Georgopolous et al., 1992, Science, 258:808-812; Hahm et al., 1994, Mol. Cell Biol., 14:7111-7123; Molnar and Georgopolous, 1994, Mol. Cell Biol., 14: 8292-8303; Wang et al., 1996, Immunity, 5:537-549; Winandy et al., 1995, Cell, 83:289-299; Molnar et al., 1996, J. of Immunol., 156:585-592; Sun et al., 1996, EMBO J., 15:5358-5369; Hansen et al., 1997, Eur. J. Immunol, 27:3049-3058; Georgopolous et al., 1997, Ann. Rev. Immunol., 15:155-176; Brown et al., 1997, Cell, 91:845-854; and Klug et al., 1998, Proc. Natl. Acad. Sci. USA, 95:657-662. 
Only three of the eight Ikaros isoforms (Ik-1, Ik-2, and Ik-3), however, contain the requisite three or more amino (N)-terminal zinc fingers that confer high affinity binding to a Ikaros-specific core DNA sequence motif in the promoters of target genes2. The formation of homo- and heterodimers among the DNA binding isoforms increases their affinity for DNA, whereas heterodimers between the DNA binding isoforms (Ik-1, Ik-2, and Ik-3) and non-DNA binding isoforms (Ik-4, Ik-5, Ik-6, Ik-7, and Ik-8) are unable to bind DNA. Therefore, non-DNA-binding Ikaros proteins with fewer than three N-terminal zinc fingers can interfere with the activity of Ikaros isoforms that can bind DNA3.
2 Sun et al., 1996, EMBO J., 15:5358-5369 
3 Molnar et al., 1996, J. Immunol., 156:585-592; and Sun et al., 1996, EMBO J., 15:5358-5369 
In mice, absence of the normal Ikaros gene results in an early and complete arrest in the development of all lymphoid lineages during both fetal and adult hematopoiesis4. Ikaros-deficient mice have a rudimentary thymus, lack peripheral lymph nodes and are characterized by a complete absence of lymphocyte progenitor cells as well as mature B-lymphocytes, T-lymphocytes, and natural killer cells. Ikaros also has a very important leukemia suppressor function which depends on its DNA binding ability: Mice heterozygous for a germline mutation which results in loss of critical DNA-binding zinc fingers of Ikaros develop a very aggressive form of lymphoblastic leukemia with a concomitant loss of heterozygosity between three and six months after birth5. Moreover, Ikaros has been localized to centromeric heterochromatin in immature lymphocyte precursors and it has been proposed that Ikaros might play an important role in recruitment and centromere-associated silencing of potentially xe2x80x9cleukemogenicxe2x80x9d growth regulatory genes.6 
4 Georgopolous et al., 1994, Cell, 79:143-156 
5 Winandy et al., 1995, Cell, 83:289-299 
6 Brown et al., 1997, Cell, 91:845-854; and Klug et al., 1998, Proc. Natl. Acad. Sci. USA, 95:657-662 
Specific molecular defects in the Ikaros gene and its encoded protein have not been previously identifed, nor has Ikaros or any of its isoforms been implicated in human disease. Determination of such defects and the correlation of the specific defect to human leukemic disease would provide particularly useful diagnostic and therapeutic tools.
The present invention provides diagnostic and therapeutic tools based on the discovery of a direct correlation of non-DNA-binding IKAROS isoforms and/or specific IKAROS gene mutations and mutant proteins with lymphoid disease, and particularly with cancer, such as leukemia. Specific Ikaros mutations resulting from splice variants which lead to an in-frame deletion of ten amino acids (xcex94KSSMPQKFLG [SEQ ID NO: 13]) upstream of the transcription activation domain and adjacent to the carboxy-terminal zinc fingers have been identified in children and infants with acute lymphoblastic leukemia (ALL), expressing high levels of dysfunctional dominant-negative Ikaros isoforms. In addition, a second specific Ikaros mutation leading to an in-frame insertion of 20 amino acids TYGADDFRDFHAIIPKSFSR [SEQ ID NO: 11] has also been identified in leukemic cells.
The identification of these specific defects and their association with ALL, as well as the correlation of dominant-negative Ikaros isoforms with hematoloic malignancy, provide useful tools for the diagnosis and monitoring of cancer, and particularly hematologic malignancy, including lymphoid malignancy and lymphoma. Such diagnostic tools correlate the abundance of dominant-negative Ikaros isoforms (non-DNA-binding isoforms) and/or the presence of specific Ikaros mutations with hematologic cell abnormality, including malignancies. The correlation of these defects in Ikaros expression in abnormal cells, such as leukemic cells, also provides thereapeutic tools for repairing the defect and restoring normal hematologic cell function.
Accordingly, the present invention provides nucleic acid and protein sequences of specific Ikaros mutations. The invention further provides methods for the analysis of Ikaros proteins and for discriminating between wild type and mutant forms, as well as between DNA-binding and non-binding isoforms.
Diagnostic methods of the invention correlate the abundance of non-DNA-binding forms of Ikaros, for example, present in a ratio  greater than 1, with disease, particularly with cancer. An abundance of non-DNA-binding isoforms and/or mutants correlates with lymphoid disease, and most particularly with leukemias, including AML, ALL, and secondary leukemias. Ikaros proteins, including isoforms and mutants, and nucleic acid sequences encoding them, can be analyzed by one or more methods described in the detailed description and examples below.
The present invention also provides for the replacement of DNA-binding forms of Ikaros in the treatment of disease, for example in the treatment of cancer such as leukemia, where DNA-binding forms are diminished or absent.